This invention relates to polyamides that bind to predetermined sequences in the minor groove of double stranded DNA that are useful for diagnosis and treatment of diseases associated with gene transcription. This invention is related to modulation of cellular or viral gene expression required for maintenance and replication of pathogens in infectious disease, such as HIV-1 and CMV. This invention is also related to modulation of cellular gene expression in non-infectious disease conditions, such as cancers involving oncogenes, e.g., her-2/neu.
Gene Therapy Approaches for HIV:
Considerable effort has been expended over the past decade to devise methods to interfere with HIV-1 gene expression in living cells in the hope that therapeutic strategies will come from these studies (recently reviewed in Kohn, D. B. and N. Sarver, Gene therapy for HIV-1 infection, in Antiviral Chemotherapy, J. Mills, P. A. Volberding, and L. Corey, Editors. 1996, Plenum Press: New York. p. 421-427.). One approach includes interference with the translation of messenger RNA into protein by the introduction of antisense oligonucleotides into lymphoid cells, as discussed in Kohn, D. B. and N. Sarver, Gene therapy for HIV-1 infection, in Antiviral Chemotherapy, J. Mills, P. A. Volberding, and L. Corey, Editors. 1996, Plenum Press: New York. p. 421-427; Bordier, B., et al., Proc. Natl. Acad. Sci. U.S.A., 92: 9383-9387 (1995) and Lisziewicz, J., et al., Proc. Natl. Acad. Sci. U.S.A., 91: 7942-7946 (1994).
Another approach involves ribozyme-mediated destruction of specific regions of HIV-1 mRNA. See Sun, L. Q., et al., Proc. Natl. Acad. Sci. U.S.A., 92: 7272-7276 (1995); Yamada, O., et al., J. Virol., 70: 1596-1601 (1996) and Zhou, C., et al., Gene, 149: 33-39 (1994). Decoy molecules, corresponding to HIV-1 RNA domains that bind regulatory proteins required for the HIV-1 life cycle (TAR RNA which binds Tat or the Rev-response element) have been used as inhibitors of HIV-1 replication (Sullenger, B. A., et al., Cell, 63: 601-608 (1990). In addition, trans-dominant mutant versions of these regulatory proteins, introduced into cells with retroviral expression vectors, have been shown to inhibit HIV-1 replication (Bevec, D., et al., Proc. Natl. Acad. Sci. U.S.A., 89: 9870-9874, 1992.).
Other approaches for direct inhibition of gene transcription, including designed or selected zinc finger peptides that recognize pre-determined DNA sequences, are described in Wu, H., et al., Proc. Natl. Acad. Sci. U.S.A., 92: 344-348 (1995) and Thiesen, H.-J., Gene Expr., 5: 229-243.(1996). DNA-cleaving ribozymes have also been tried (Raillard, S. A. and G. F. Joyce, Biochemistry, 35: 11693-11701(1996)). Triple helix-forming oligonucleotides have been used to block HIV-1 integration: Bouziane, M., et al., J. Biol. Chem., 271: 10359-10364 (1996). Triple helix-forming oligonucleotides have also been used specifically cleave HIV-1 DNA with a metalloporphyrin group attached to the oligonucleotide, as described by Bigey, P., G. Pratviel, and B. Meunier, Nucleic Acids Res., 23: 3894-3900 (1995). Additionally, the DNA-binding calicheamicin oligosaccharides have the potential for use in anti-HIV-1 therapy but have not as yet been applied to this disease. See Ho, S. N., et al., Proc. Natl. Acad. Sci., 91: 9203-9307 (1994) and Liu, C., et al., Biochemistry, 93: 940-944 (1996).
For any gene therapy approach to be successful, several criteria must be met by the therapeutic agent: First, tile agent must not possess any general cell toxicity and should not elicit an immune response. Second, the agent must be cell-permeable or amenable to viral delivery and, in the case of the DNA-binding agents, the therapeutic agent must transit to the nucleus and bind the target sequence with high affinity and specificity in the context of cellular chromatin. Third, binding of the agent to its DNA or RNA target sequence must interfere with gene transcription or protein translation.
Each of the potential approaches listed above has its own unique advantages and limitations. For example, while nucleic acid-based approaches (antisense, decoy and triple helix-forming oligonucleotides and ribozymes) have the potential for sequence selectivity and can effectively inhibit transcription or translation in vitro, these molecules suffer from poor cell permeability and other delivery systems, such as retroviral vectors in the case of the ribozymes (Zhou, C., et al., 1994) or liposomes or other delivery strategies in the case of antisense or triple helix oligonucleotides, must be used for effective gene inhibition (reviewed in Kohn and Sarver, 1996). Similarly, zinc finger peptides must be introduced via a gene therapy approach with an appropriate viral expression vector since these peptides cannot directly enter cells. See Choo, Y., et al., Nature, 372: 642-645 (1994).
One additional problem with gene therapy approaches is that they must be performed on lymphoid cells ex vivo and, once an xe2x80x9cHIV-protectedxe2x80x9d cell population is established, these cells must then be introduced into the patient.
In contrast to gene therapy approaches, HIV protease inhibitors taken in combination with standard anti-retroviral agents (AZT) have recently shown success in clinical trials. Wei, X. et al., Nature, 373: 117-122 (1995); Ho, D. D. et al., Nature, 373: 123-126 (1995).
The key to the anti-HIV properties of these drugs is that they strike at two separate phases of the virus life cycle, limiting the ability of spontaneous mutations to result in inhibitor-resistant strains of the virus. Small molecule inhibitors of HIV-1 RNA transcription which would target a third phase of the virus life cycle would be highly desirable. Cell-permeable sequence-specific DNA-binding ligands would circumvent the problems associated with other forms of gene therapy and could compliment the protease inhibitor-anti-retroviral agent cocktail approach mentioned above. The calicheamicin oligosaccharides satisfy some of the requirements for a therapeutic agent; these molecules are sufficiently hydrophobic to pass through cell membranes but these molecules possess severely limited sequence specificity (4 base pairs) and bind DNA with very low affinities (100 xcexcM or higher required for inhibition of protein-DNA interactions . See Ho, S. N., et al., Proc. Natl. Acad. Sci., 91: 9203-9307 (1994) and Liu, C., et al., Biochemistry, 93: 940-944 (1996)).
Thus, new classes of cell-permeable molecules that possess higher degrees of DNA sequence specificity and affinity are needed for the treatment of AIDS and other infectious diseases. We describe below the successful development of a new class of highly specific designed small molecule ligands with great potential for inhibition of HIV-1 gene transcription.
The HIV-1 Enhancer and Promoter:
A recent review has summarized our current knowledge of the protein factors required for the control of RNA initiation and elongation by RNA polymerase II at the HIV-1 promoter (Jones, K. A. and B. M. Peterlin. 1994. Control of RNA initiation and elongation at the HIV-1 promoter. Annu. Rev. Biochem., 63: 717-743). Thus only those aspects of HIV-1 transcription that relate to transcription inhibition are discussed herein. For HIV, the template for synthesis of both new viral RNA and messenger RNA (for viral protein synthesis) is the integrated provirus, the product of reverse transcription of the viral RNA in the infected cell. HIV-1 utilizes the transcription machinery of the host cell but encodes its own trans-activators, Tat and Rev, that are responsible for RNA elongation and utilization. The HIV-1 promoter is located in the U3 region of the leftward (5xe2x80x2) long terminal repeat (see FIG. 11 below, taken from Jones and Peterlin, 1994). The core promoter and enhancer elements span a region of approximately 250 base pairs and include TATA and initiator elements and the binding sites for the following cellular transcription factors: Sp1, NF-xcexaB, LEF-1, Ets-1 and USF. Sequences upstream of the NF-xcexaB sites contribute only marginally to HIV-1 promoter activity either in vitro or in transfected cell lymphoid cell lines. Waterman, M. L. and K. A. Jones, New Biologist, 2: 621-636 (1990). However, these upstream sequences, and presumably the protein factors which bind these upstream sequences, are important for viral replication, and hence promoter activity, in peripheral blood lymphocytes and in some T cell lines. Kim, J., et al., J. Virol, 67: 1658-1662 (1993).
Two of the binding sites in the upstream region correspond to recognition sites for activator proteins that are lymphoid cell specific (or highly enriched in T cells) and are shared with the promoter of the T cell receptor (TCRxcex1) gene: these are the Ets-1 and LEF-1 transcription factors. The essential role of the upstream region has recently been reproduced in vitro with a chromatin reconstitution assay (Sheridan, P. L., et al., Genes Dev., 9: 2090-2104 (1995)).
Packaging of the HIV-1 promoter into nucleosomes strongly repressed transcription, but this repression could be relieved by pre-incubation of the template with the HIV-1 enhancer-binding proteins, LEF-1 and Ets-1. LEF-1 and Ets-1 thus apparently act in concert to prevent nucleosome-mediated repression in vivo. Inhibition of formation of this complex by small molecule inhibitors could well represent a viable target for HIV-1 gene therapy. LEF-1 is a member of the HMG family of proteins and binds DNA as a monomer. DNA binding is in the minor groove and results in a large distortion of the DNA helix (unwinding and bending) (Love, J. J., et al., Nature, 376: 791-795 (1995)).
In addition to acting as an architectural transcription factor, LEF-1 possesses a strong trans-activation domain which can function when artificiality transferred to other DNA-binding proteins (Giese, K., et al., Genes Dev., 9: 995-1008 (1995)). This region of the HIV-1 enhancer might thus prove to be an effective target for inhibition of viral transcription and hence virus replication.
The HIV-1 promoter also contains tandem binding sites for NF-xcexaB, a factor that is strongly induced by HIV infection (Bachelerie, F., et al. Nature, 350: 709-712 (1991)) and multiple binding sites for the general transcription factor Sp1. The mechanisms of NF-xcexaB activation have been reviewed by Jones and Peterlin, 1994. Important for this discussion, NF-xcexaB has been shown to contact both Sp1 and the TBP subunit of the basal transcription factor TFIID. Perkins, N. D., et al., Mol. Cell. Biol., 14: 6570-6583 (1994). Additionally, Sp1 has been shown to interact with the TAF110 subunit of TFIID (110 kDa TBP-associated factor) (Chen, J. L., et al., Cell, 79: 93-105, 1994). It is the binding of TFIID via the TBP interaction with the TATA element that nucleates the assembly of the complete RNA polymerase II transcription complex (reviewed in Maldonado, E. and D. Reinberg, Current Opinion in Cell Biology, 7: 352-361. 1995). Thus NF-xcexaB may function through recruitment of Sp1 and TFIID to the HIV-1 promotor via these protein-protein interactions. Thus blocking the NF-xcexaB-DNA and/or Sp1-DNA interaction is another potential target for HIV therapy. Since these factors, and especially Sp1 and TFIID, are utilized by a wide range of cellular genes, the binding sites for these factors would not be good targets for HIV-specific inhibition (or any gene-specific inhibition). However, the sequences adjacent to these sites, that are unique to HIV-1 proviral DNA, are excellent candidate targets for the design of inhibitory DNA ligands (see below).
Polyamide DNA-binding Ligands:
Simple rules have been developed to rationally determine the sequence-specificity of minor-groove-binding polyamidecontaining N-methylimidazole and N-methylpyrrole amino acids. These synthetic pyrrole-imidazole polyamides bind DNA with excellent specificity and very high affinities, even exceeding the affinities of many sequence-specific transcription factors (Trauger et al., Nature, 382: 559-561, 1996). An Im/Py pair distinguishes G.C from C.G and both of these from A.T or T.A base pairs. Wade, W. S., et al. describes the design of peptides that bind in the minor groove of DNA at 5xe2x80x2-WGWCW-3xe2x80x2 sequences (where W is either A or T, and a W.W pairs is an A.T or a T.A base pairs by a dimeric side-by-side motif. J Am. Chem. Soc. 114, 8783-8794 (1992); Mrksich, M. et al. describes antiparallel side-by-side motif for sequence specific-recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamidenetropsin. Proc. Natl. Acad. Sci. USA 89, 7586-7590 (1992); Trauger, J. W., et al., describes the recognition of DNA by designed ligands at subnanomolar concentrations. Nature 382, 559-561 (1996). A Py/Py pair specifies A.T from G.C but does not distinguish A.T from T.A. Pelton, J. G. and Wemmer, D.E. describes the structural characterization of a 2-1 distamycin A-d(CGCAAATTTGGC)(SEQ ID NO:16) complex by two-dimensional NMR. Proc. Natl. Acad Sci. USA 86, 5723-5727 (1989); White, S., et al. Biochemistry 35, 12532-12537 (1996) describes the effects of the A.T/T.A degeneracy of pyrrole-imidazole polyamide recognition in the minor groove of DNA, the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides, and also describes the 5xe2x80x2-3xe2x80x2 N-C orientation preference for polyamide binding in the minor groove.
It has been found that a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp) when incorporated into a polyamide and paired opposite Py, provides the means to discriminate A.T from T.A. White S., et al., Nature 391 436-438 (1998). Unexpectedly, the replacement of a single hydrogen atom on the pyrrole with a hydroxy group in a Hp/Py pair regulates the affinity and the specificity of a polyamide by an order of magnitude. Utilizing Hp together with Py and Im in polyamides to form four aromatic amino acid pairs (Im/Py, Py/Im, Hp/Py, and Py/Hp) provides a code to distinguish all four Watson-Crick base pairs in the minor groove of DNA.
The preferred corresponding designed specific polyamides resulting from this invention are of the form
X1X2 . . . Xmxe2x88x92xcex3xe2x88x92X(m+1) . . . X(2mxe2x88x921)X2m-xcex2-Dp
wherein X1, X2, Xm, X(m+1), X(2mxe2x88x921), and X2m are carboxamide residues forming carboxamide binding pairs X1/X2m, X2/X(2mxe2x88x921), Xm/X(m+1), and xcex3 is xcex3-aminobuytic acid or 2,4 diaminobutyric acid and Dp is dimethylaminopropylamide, and where
carboxamide binding pair X1/X2m corresponds to base pair N1.Nxe2x80x21,
carboxamide binding pair X2/X(2mxe2x88x921) corresponds to base pair N2.Nxe2x80x22,
carboxamide binding pair Xm/X(m+1) corresponds to base pair Nm.Nxe2x80x2m.
In general, the specific polyamide DNA-binding ligands were designed by using a method that comprises the steps of identifying the target DNA sequence 5xe2x80x2-WN1N2 . . . NmW-3xe2x80x2; representing the identified sequence as 5xe2x80x2-Wab . . . xW-3xe2x80x2, wherein a is a first nucleotide to be bound by the X1 carboxamide residue, b is a second nucleotide to be bound by the X2 carboxamide residue, and x is the corresponding nucleotide to be bound by the Xm carboxamide residue; defining a as A, G, C, or T to correspond to the first nucleotide to be bound by a carboxamide residue in the identified six base pair sequence.
Carboxamide residues were selected sequentially as follows: Im was selected as the X1 carboxamide residue and Py as the X2m carboxamide residue if a was G. Py was selected as the X1 carboxamide residue and Im as the X2m carboxamide residue if a was C. Hp was selected as the X1 carboxamide residue and Py as the X2m carboxamide residue if a was T. Py was selected as the X1 carboxamide residue and Hp as the X2m carboxamide residue if a was A.
The remaining carboxamide residues were selected in the same fashion. Im was selected as the X2 carboxamide residue and Py as the X2mxe2x88x921 carboxamide residue if b was G. Py was selected as the X2 carboxamide residue and Im as the X2mxe2x88x921 carboxamide residue if b was C. Hp was selected as the X2 carboxamide residue and Py as the X2mxe2x88x921 carboxamide residue if b was T. Py was selected as the X2 carboxamide residue and Hp as the X2mxe2x88x921 carboxamide residue if b was A.
The selection of carboxamide residues was continued through m iterations. In the last iteration, Im was selected as the Xm carboxamide residue and Py as the Xm+1 carboxamide residue if x was G. Py was selected as the Xm carboxamide residue and Im as the Xm+1 carboxamide residue if x was C. Hp was selected as the Xm carboxamide residue and Py as the Xm+1 carboxamide residue if x was T. Py was selected as the Xm carboxamide residue and Hp as the Xm+1 carboxamide residue if x was A.
In one preferred embodiment, the polyamide includes at least four consecutive carboxamide pairs for binding to at least four base pairs in a duplex DNA sequence. In another preferred embodiment, the polyamide includes at least five consecutive carboxamide pairs for binding to at least five base pairs in a duplex DNA sequence. In yet another preferred embodiment, the polyamide includes at least six consecutive carboxamide pairs for binding to at least six base pairs in a duplex DNA sequence. In one preferred embodiment, the improved polyamides have four carboxamide binding pairs that will distinguish A.T, T.A, C.G and G.C base pairs in the minor groove of a duplex DNA sequence.
DNA target sequence recognition thus depends on a code of side-by-carboxamide residue pairings in the minor groove of double stranded DNA. These compounds represent the only class of synthetic small molecules that can bind predetermined DNA sequences with affinities and specificities comparable to DNA-binding proteins.
This invention provides specific polyamides that are useful for modulating the expression of a cellular or viral gene by binding to predetermined target sequences adjacent to the binding site of a transcription factor protein in the minor groove of double stranded DNA. Suitable cellular genes include both eukaryotic and prokaryotic genes. The cellular gene can be present in the original native cells, in cells transfected or transformed with a recombinant DNA construct comprising the cellular gene or in an in vitro in a cell-free system. The viral gene can be present in a cell or in an in vitro in a cell-free system.
The polyamides of the present invention can act as specific inhibitors of gene transcription in vivo or in vitro as therapeutic agents in disease conditions related to the transcription of at least one cellular or viral gene. Studies with three accepted model systems have shown that polyamides do interfere with the binding of sequence-specific minor groove transcription factor proteins as well as with components of the basal transcription machinery and thus block transcription of target genes.
Hereinafter, N-methylpyrrole carboxamide may be referred to as xe2x80x9cPyxe2x80x9d, N-methylimidazole carboxamide may be referred to as xe2x80x9cImxe2x80x9d, 3-hydroxy-N-methylpyrrole carboxamide may be referred to as xe2x80x9cHpxe2x80x9d, xcex3-aminobutyric acid may referred to as xe2x80x9cxcex3xe2x80x9d, xcex2-alanine may be referred to as xe2x80x9cxcex2xe2x80x9d, glycine may be referred to as xe2x80x9cGxe2x80x9d, dimethylaminopropylamide may be referred to as xe2x80x9cDpxe2x80x9d, and ethylenediaminetetraacetic acid may be referred to as xe2x80x9cEDTAxe2x80x9d.
The invention encompasses polyamides having xcex3-aminobutyric acid or a substituted xcex3-aminobutyric acid to form a hairpin with a member of each carboxamide pairing on each side of it. Preferably the substituted xcex3-aminobutyric acid is a chiral substituted xcex3-aminobutyric acid such as (R)-2,4-diaminobutyric acid. In addition, the polyamides may contain an aliphatic amino acid residue, preferably a xcex2-alanine residue, in place of a Hp or Py carboxamide. The xcex2-alanine residue is represented in formulas as xcex2. The xcex2-alanine residue becomes a member of a carboxamide binding pair. The invention further includes the substitution as a xcex2/xcex2 binding pair for non-Im containing binding pair. Thus, binding pairs in addition to the Im/Py, Py/Im, Hp/Py and Py/Hp are Im/xcex2, xcex2/Im, Py/xcex2, xcex2/Py, Hp/xcex2, xcex2/Hp, and xcex2/xcex2.
The polyamides of the invention can have additional moieties attached covalently to the polyamide. Preferably the additional moieties are attached as substituents at the amino terminus of the polyamide, the carboxy terminus of the polyamide, or at a chiral (R)-2,4-diaminobutyric acid residue. Suitable additional moieties include a detectable labeling group such as a dye, biotin or a hapten. Other suitable additional moieties are DNA reactive moieties that provide for sequence specific cleavage of the duplex DNA.
A central aspect of the present invention is the use of unique or rare sequences adjacent to the binding sites for common transcription factors as the target sequences for the design of polyamides. It has been found that (1) sequences adjacent to the binding sites for required transcription factors are unique to the genes under study and are not found in other genes in the current nucleic acid data bases; (2) polyamides targeted to these sequences are effective inhibitors of protein-DNA interactions; (3) such polyamides are inhibitors of transcription factor-dependent gene transcription in vitro; and (4) the polyamides are cell permeable agents and have been shown to inhibit transcription of target genes in human cells in culture.
Most importantly, several designed polyamides have been shown to inhibit transcription of specific genes in vivo and thus these compounds must be both cell permeable and once inside the cell they must be able to transit the nuclear envelope and bind their target sites within chromatin (Gottesfeld, J. M., et al., Nature, 387: 202-205, 1997). These results demonstrate that designed pyrrole-imidazole polyamides are useful in the treatment of diseases, particularly viral diseases, including AIDS, as well as many other diseases for which specific candidate gene targets have been identified.
The present invention provides specific polyamides which inhibit the transcription of DNA upstream or downstream of transcriptional factors such as the 5S RNA gene transcriptional factor TFIIIA, the minor groove-binding protein TATA-box binding protein (TBP), Ets-1 and the lymphold enhancer factor LEF-1 protein. These polyamides act as gene-specific inhibitors of transcription since these polyamides are selective for the sequences flanking these protein binding sites that are, in turn, gene-specific. The polyamides are useful as therapeutics for the treatment of cancer as well as for the treatment of diseases caused by viruses and other pathogens (such as bacterial, fungal, etc.)
The present invention provides a composition comprising a transcription inhibiting amount of at least one polyamide chosen from the group consisting of ImPyPyPy-xcex3-ImPyPy-xcex2-Dp, ImPy-xcex2-ImPy-xcex3-ImPy-xcex2-ImPy-xcex2-Dp and mixtures thereof and a pharmaceutically acceptable excipient suitable for the treatment of HIV-1 infection. The invention also provides a method of treating a human patient with an HIV-1 infection comprising the step of administering a composition comprising a transcription inhibiting amount of at least one polyamide chosen from the group consisting of ImPyPyPy-xcex3-ImPyPy-xcex2-Dp, ImPy-xcex2-ImPy-xcex3-ImPy-xcex2-ImPy-xcex2-Dp and mixtures thereof and a pharmaceutically acceptable excipient. Preferably a transcription inhibiting amount corresponds to an extracellular concentration of polyamide of about 100 nanomolar to about 10 micromolar. In one preferred embodiment, a transcription inhibiting amount corresponds to an extracellular concentration of about one micromolar to about ten micromolar ImPyPyPy-xcex3-ImPyPy-xcex2-Dp mixed with about one micromolar to about ten micromolar ImPy-xcex2-ImPy-xcex3-ImPy-xcex2-ImPy-xcex2-Dp.
The present invention provides methods of treating cells in vitro as well as treating a human patient or a non-human organism in vivo. In one preferred embodiment, the invention provides a method of treating HIV-1 infected human blood cells in vitro comprising the step of administering a composition comprising a transcription inhibiting amount of at least one polyamide chosen from the group consisting of ImPyPyPy-xcex3-ImPyPy-xcex2-Dp, ImPy-xcex2-ImPy-xcex3-ImPy-xcex2-ImPy-xcex2-Dp and mixtures thereof.
In other embodiments, the invention provides a diagnostic kit for detecting the identified target DNA sequence by employing the selective polyamides and a suitable system for the detection of the polyamide bound to the DNA.